Disinfecting preparation containing chlorine in alcohol

ABSTRACT

Liquid germicidal formulation containing, as the active ingredients, a combination of an alcohol and an organic N-chloroamine, which combination shows a high bactericidal activity and an unexpected stability in time, while maintaining its effectiveness substantially unchanged even after a prolonged storage. The formulation comprises, in water, chloramine-T or chloramine-B and one or more, aliphatic alcohols having up to 12 carbon atoms, preferably isopropanol, said solution having a pH not lower than 8.5.

SPECIFICATION

[0001] The present invention concerns a disinfecting preparationcontaining chlorine in alcohol. More specifically, the invention relatesto a liquid germicidal formulation containing, as the activeingredients, a combination of an alcohol and an organic N-chloroamine,which combination shows a high bactericidal activity and an unexpectedstability in time, while maintaining its effectiveness substantiallyunchanged even after a prolonged storage.

[0002] The bactericidal properties of alcohols, known since ancienttimes, have been studied on a scientific basis starting from thebeginning of this century. Some of such products, firstly ethyl alcoholand isopropyl alcohol, have reached in the field of disinfectants aquite remarkable diffusion, also in view of the advantages connectedwith their water solubility, with the ease of evaporation and with theirreduced toxicity. Actually, some higher aliphatic alcohols are moreeffective than the lower ones as antimicrobial agents, the highesteffectiveness being reached by alcohols having 6-8 carbon atoms.However, the low volatility and the unpleasant odor of these compoundshave greatly limited their use. Also some aromatic alcohols find somelimited application as disinfectants, among which benzyl alcohol andphenethyl alcohol. The latter, however, suffer from the disadvantage ofbeing more toxic.

[0003] Ethanol shows its maximum potency as a 60-75% aqueous solution(weight percentage), while solutions of lower or even higherconcentration take a longer time to exert the same germicidal effects.Both alcohols with three carbon atoms, i.e. n-propanol and isopropanol,show a higher activity than ethanol, with a maximum level atconcentrations around 60% by weight. In spite of their unquestionedantibacterial activity, from the point of view of the activity spectrumaliphatic alcohols have the drawback of being totally ineffectiveagainst spore-forming micro-organisms.

[0004] Another product the use of which as disinfectant is wellestablished since the last century is chlorine. Chlorine was employed,in the form of calcium chloride, for treating the sewage of the city ofLondon in the mid-XIX century. In the same period, chlorine was used asa disinfectant in the hospital of Dr. Semmelweis in Vienna, to fight thepuerperal fever. Although the mechanism of action of chlorine has notyet been fully clarified, it is believed that chlorine performs itsdisinfecting action by releasing in water hypochloric acid (HClO), whichis responsible for the destruction of microorganisms. The concentrationsof active chlorine required to kill most of the bacterial species may beof the order of 1 ppm, while higher concentrations are normally requiredto destroy spores and mycobacteria.

[0005] In the current practice, the term chlorine disinfectant isgenerically employed to refer to any disinfectant consisting in anaqueous solution of chlorine, hypochlorite or hypochlorous acid andalso, in many cases, to other organic or inorganic chlorine-releasingcompounds, such as, e.g., chloroamines (ClNH₂, Cl₂NH, Cl₃N),N-chlorosulfonamides (e.g., sodium N-chloro-p-toluenesulfonamide orchloramine-T, and sodium N-chlorobenzenesulfonamide or chloramine-B),N-chloroisocyanuric acids. Such compounds are also employed, at the sametime, in view of their activity as chemical oxidants, and areconsequently the most widely used products for the treatment of drinkingwater, for sanitizing swimming pools and for water treatment in the foodindustry.

[0006] In view of the foregoing, the interest in formulating adisinfectant containing, as the active ingredients, both alcohol andchlorine is quite clear. A similar preparation would join the antisepticproperties typical of each one of these two classes of compounds,thereby resulting in a product with an enhanced potency and a wideractivity spectrum. Although the known art includes many disinfectantsthat combine the antibacterial activity of alcohols with the activity ofother agents, such as, e.g., iodine, phenols or chlorhexidine, nopreparations consisting of combinations of chlorine and alcohol appearto be presently on the market. This is apparently due to the strongoxidizing action exerted by chlorine on such organic products, that areknown to be relatively easily oxidizable.

[0007] As it is known, the power of a compound as an oxidant is measuredon one hand by thermodynamic factors, in particular by the standardoxidation potential of the compound, and on the other hand by kineticfactors, which govern the rate of the oxidation reaction. As a matter offact, although in the absence of information on the reaction kinetics itis impossible to precisely foresee the performance of an oxidizingsystem, a first valuable indication is always found in the standardoxidation potentials. It is, actually, the high standard oxidationpotential for the reduction of molecular chlorine to chloride ion(Cl₂+2e⁻

2Cl⁻) and that for the reduction of hypochloric acid to chloride ion(HOCl+H⁺+2e⁻

Cl⁻+H₂O)— respectively, E₀=1.36 V and E₀=1.49 V—that give the mostimmediate indication of the potency of the chlorine-based agents aschemical oxidants (see, e.g., R. G. Rice and M. Gomez-Taylor,Environmental Health Perspectives, 69, 31-44, (1986)).

[0008] In this connection, it has been shown experimentally (D. Coatesand J. E. Death, Journal of Clinical Pathology, 31, 148-152, (1978))that mixtures of various alcohols or glycols (i.e., methanol,n-propanol, isopropanol, ethanol and ethanediol) in aqueous solution atvarious concentrations (from 10 to 50% by weight) with sodiumhypochlorite at a level of available chlorine of 2000 ppm, althoughhaving an interesting sporicidal activity when freshly prepared, hadlost practically all available chlorine after few hours. In order toexploit the advantageous biological activity discovered, therefore, theconcerned publication suggests to store the solutions of alcohol andhypochlorite in separate containers, and to mix them just before use.

[0009] A first measure aimed at limiting as much as possible thereactivity between alcohols and chlorine disinfectants may be to use, ina possible preparation, a chlorine source characterized by an oxidationpotential lower than sodium hypochlorite, which is able to supplychlorine much more slowly than the common hypochlorites. Productsparticularly suitable to this end are the chloroamines, as it may beinferred from the following table. The table shows the standardoxidation potentials at 25° C. in aqueous solution (according to twodifferent literature references, i.e. a): R. G. Rice and M.Gomez-Taylor, loc. cit.; b): Ullmann's Encyclopedia of IndustrialChemistry, 5^(th) ed., VCH Verlagsellschaft A 28, 87 (1996)). E₀,V-Ref.^(a)) E₀, V-Ref.^(b)) Chlorine 1.36 1.36 Hypochlorous acid 1.491.50 Hypochlorite ion — 0.90 Chlorine dioxide 1.275 1.71 Monoohloroamine1.16 0.75

[0010] Although they are not as powerful as chlorine, chloroamines havethe advantage of being more stable, since they are less rapidlyreactive. In water, chloroamines slowly hydrolyze, generatinghypochloric acid according to the general reaction RR′NCl+H₂O

RR′NH+HOCl. In view of the fact that the bactericidal activity of thesecompounds is due to the release of hypochloric acid, the value of theequilibrium constant of the above reaction is used to express theactivity of chloroamines as disinfectants. Since hypochloric acid isreleased gradually from these compounds, the latter may keep theirdisinfecting activity for longer periods of time, compared tohypochlorites.

[0011] Among the possible organic and inorganic compounds belonging tothe family of chloroamines, the present invention is concerned with twospecific agents, already mentioned in the foregoing, consisting in themonosodium salts of two N-chlorosulfonamides, i.e.N-chloro-p-toluenesulfonamide sodium salt or chloramine-T,

[0012] and N-chlorobenzenesulfonamide sodium salt or chloramine-B

[0013] Both these compounds are known as topical antiseptics and forwater sterilization, as they are active against a wide spectrum ofmicroorganisms As

[0014] EP 372415 disclosed a process for the stabilisation ofChloramine-T and -B solutions, according to which oxidation-resistantbuffers (HEPPS and BICIN) are used at pH between 7 and 12.

[0015] U.S. Pat. No. 3,767,586 discloses a process for preparing stablesolutions of N-halo compounds by reaction of a N-hydrogen compound, ahalogen, an alkali or alkaline earth metal hydroxide, the presence of abuffer at pH 4.5-8.5.

[0016] Block, S. S., “Disinfection, Sterilisation and PreservationFourth Edition” (Pg. 143), Lea & Febiger, Philadelphia, US, describesinorganic chloramines and their use as bactericidal agents, includingchloramine-T, whose bactericidal action is obtained at low pH and withlong exposure. for all of the other chlorine disinfectants, the use ofchloramine-T and chloramine-B in alcohol-based formulations was notconsidered to be commercially possible according to the currentpractice, in view of the oxidizing activity exerted by chlorine onalcohols.

[0017] One only known patent publication, i.e. patent No. 2255 of theformer German Democratic Republic, issued in 1943, discloses antisepticformulations which are said to contain both a chlorosulfonamide andalifatic alcohols. However, such preparations also contain an organicdye extremely prone to oxidation, e.g. naphthol yellow S or2,4,4-trinitrophenol sodium salt. According to the document, the alcoholprevents the organic dye from being oxidized by chloramine. From thecomparative tests reported in the patent, a solution of naphthol yellowdye and chloramine-T in distilled water heated at 40° C. is rapidlydegraded, as it is evidenced by the strong odor emanating from thesolution, while a similar solution wherein 50% by volume of ethylalcohol is present does not undergo any detectable odor changes. In theevent that the temperature is kept at 50° C. for 16 hours, theconcentration of active chlorine in the water solution of dye andchloramine-T is reduced by 92% by weight, while the concentration ofactive chlorine in the solution containing 50% of alcohol is reduced by4.5% by weight only. Such a reduction is in any case too marked to allowto consider a disinfecting product stable according to the currentstandards. In addition, the document does not supply any specificindication that may be used to obtain, in general, alcohol solutions ofchloramine-T or -B of any desired concentration, which are stable forquite long periods of time. On the contrary, the document seems tosuggest that any formulation based on N-chlorosulfonamide and a dye,mixed with alcohols, may behave as a stable solution (within the limitsinferrable from the example referred to above).

[0018] On the contrary, it has been found, according to the presentinvention, that a hydroalcoholic solution of chloramine-B orchloramine-T may remain stable for periods of time of several months,losing amounts of active chlorine of no more than few units per cent,provided that the pH of the solution is kept above 8.5, preferably above9.5. The critical influence of pH may be explained if one considers thatchloramines B and T, being strong electrolytes, readily dissociate inaqueous solution, thus transforming into the corresponding anionic form:

[0019] (wherein X may be a hydrogen atom or the —CH₃ group). In view ofthe markedly basic character of such species, it is expected that theattack by protons possibly present in the solution takes place quiteeasily through an acid-base equilibrium. Considering that the activespecies in the alcohol oxidation reaction is likely to be the protonatedspecies, it may be understood that the pH can exert a great influence onthe said reaction, hindering the reaction as much as the protonconcentration is low.

[0020] In order to give an experimental confirmation of the abovehypothesis, made in the frame of the invention, the following tableshows the percent amount of available chlorine measured in aqueoussolutions containing 60% by weight isopropanol and 10 g/l chloramine-B(equivalent to 2500 ppm of active chlorine), as well as variousphosphate buffers, depending on the desired pH, the solution being keptat 40° C. for 4 weeks. TABLE 1 Influence of pH on chlorine stability pH% active chlorine after 4 weeks 6.75-7.35 26 7.76-8.80 70 8.5-9.0 909.46-9.69 95 10.43-10.82 98

[0021] The data presented in the above table show that beyond a limit ofpH close to 8.5 the stability of chlorine in time drastically increases.Above the concerned limit, the oxidation-reduction reaction ofchloramine with alcohol appears to proceed extremely slowly, leavingpractically unaltered the concentration of available chlorine.

[0022] Accordingly, the present invention specifically provides adisinfecting preparation containing chlorine in alcohol solutioncomprising, in water, chloramine-T or chloramine-B as defined above, andone or more aliphatic alcohols having up to 12 carbon atoms, saidsolution having a pH not lower than 8.5. Preferably, the said desired pHvalue is maintained in the disinfecting preparation by means of asuitable buffer.

[0023] In order to perform its germicidal activity at the level normallyrequired to a disinfecting agent, the formulation according to theinvention contains said chloramine-T or said chloramine-B at aconcentration corresponding to 1000-2500 ppm of available chlorine, theoptimal concentration being around 1100-1150 ppm of available chlorine.By way of example, a composition according to the invention containing0.5% by weight of chloramine-T contains about 1120 ppm of activechlorine.

[0024] Preferably, the aliphatic alcohols are chosen from ethanol,n-propanol and isopropanol, and are present at a total concentrationcomprised between 40 and 70% by weight. It has been experimentallyascertained that the stability of chlorine in the solution according tothe invention, measured by the percentage of available chorine andreferred to the starting concentration, after storage at a fixedtemperature for a given period of time, is slightly higher forisopropanol solutions than for ethanol solutions. This finding, togetherwith the already mentioned higher bactericidal activity of isopropylalcohol compared with ethyl alcohol, makes isopropyl alcohol a preferredingredient in the disinfecting formulations according to the invention.

[0025] In addition to possessing a high disinfecting power towards awide spectrum of microorganisms and, moreover, a considerable sporicidalactivity, the proposed preparation has also the property of removing anyresidues of adhesive tape. The latter is a quite important property inview of the use of the disinfecting product in out-patient treatmentcenters, in blood collection centers and in hospital departments, andcannot be obtained with alcohol-based disinfectants. Such property is aconsequence, in the preferred embodiments of the invention, of thepresence of a borate buffer in the composition. The borate bufferperforms, on one hand, the function of maintaining the pH at the highlevels desired (as the system H₃BO₃/H₂BO₃ ⁻ has a pK of about 9.2 at 25°C., and thus has a buffering pH between 8 and 10) and, on the otherhand, enhances the removal of any tape adhesive that may be present onthe patient's skin. It is known, actually, that borate ion can removethe residues of the rubber adhesives currently used in adhesivemedications and plasters.

[0026] The inclusion of the system boric acid/sodium hydroxide in thedisinfecting formulation according to the invention also has theadvantage of adding the two functions referred to above withoutinterfering in any way with the system chloramine-alcohol, and withoutaffecting the stability of such system. Comparative tests have evidencedthat among the other possible agents able to give a good removal of theadhesive, such as, e.g., benzyl alcohol and dichloromethane, the boratebuffer is able to assure the highest stability of chlorine. In addition,dichloromethane would bring about unacceptable toxicity problems for adisinfecting product for topical use as the one proposed herein.

[0027] The concentration range of the borate buffer that allows toobtain the maximum stability is quite limited, owing to the possibilityof precipitation of sodium borate. Precipitation may occur, inparticular, at low temperatures and in presence of an excess of sodiumions. Specifically, the optimal weight concentrations of the componentsof borate buffer in the formulation according to the invention are thefollowing: boric acid 0.153-0.155% sodium hydroxide 0.0396-0.064%. 

[0028] Thus, some particularly advantageous embodiments of the inventionhave the following parameters: chloramine-T or chloramine-B 1000-2500ppm of available chlorine ethanol or isopropanol 50-60% by weightpH >8.5. boric acid 0.153-0.155% by weight sodium hydroxide0.0396-0.064% by weight water q.s. to 100%.

[0029] Preferably, the chloramine is chloramine-T, at a concentrationcorresponding to 1100-1150 ppm of available chlorine, and the alcohol isisopropanol, at a concentration of 50% by weight. The pH of suchpreferred formulations is between 10.4 and 10.9. In order to achieve themaximum chlorine stability in the storage of the product for periods oftime of at least several months, the preparation should be packaged inopaque containers, preferably dark, in order to avoid that the oxidationof alcohol is catalyzed by the electromagnetic radiation.

[0030] Some specific embodiments of the disinfectant formulation of theinvention are described below for merely illustrative purposes, togetherwith the results of the experimental studies carried out on the saidformulation.

EXAMPLE Production of a Disinfecting Preparation Containing Chlorine inAlcohol

[0031] The production of a preferred formulation according to theinvention is carried out according to the following procedure, referredto a batch of 1000 kg of product:

[0032] 1. 396 g of sodium hydroxide is dosed in 28 kg of purified water,and complete dissolution is ascertained (solution 1);

[0033] 2. 1530 g of boric acid is dosed, dissolved in 71 kg of purifiedwater at 40-45° C., and complete dissolution is ascertained (solution2);

[0034] 3. solution 1 is joined to solution 2; 394.07 kg of purifiedwater is added and the mixture is stirred for 15 minutes;

[0035] 4. to the solution thus obtained 500 kg of isopropanol is addedslowly, while stirring;

[0036] 5. 5 kg of chloramine-T is added, and the mixture is stirred for30 minutes, and finally filtered with a 1 μm filter.

[0037] The product thus obtained may be stored at room temperature forat least eighteen months without undergoing any appreciable loss ofactive chlorine. In the event that a commercial product with shelf lifeof several years is desired, the bottle may be packaged with a suitabledosing cap, wherein the prescribed amount of chloramine is kept separatefrom the hydroalcoholic solution. Just before opening the bottle, byacting on the dosing cap the two components are contacted with eachother and mixed, ad the final desired formulation is obtained. Theproduct thus obtained may be kept in the mixed state for at leasteighteen months.

[0038] Stability tests

[0039] With the preparation obtained according to the above procedure,having the following composition (percentage by weight): chloramine-T 0.5% (1120 ppm of active chlorine) isopropanol   50% boric acid 0.153%sodium hydroxide 0.0396%  purified water q.s. to 100%

[0040] and having a pH of 10.4-10.9, stability tests at room temperaturewere carried out, in order to ascertain the possibility of storing theproduct with no appreciable alterations for at least eighteen months.The results of such tests for two different batches of product are setforth in the following table. TABLE 2 Long term stability at roomtemperature Batch A Batch B Time active chlorine alcohol active chlorinealcohol (months) (ppm) (%) (%) pH (ppm) (%) (%) pH 0 1120 100.0 49.910.76 1120 100.0 50.2 10.88 1 1113 99.4 50.0 10.81 1120 97.5 50.2 10.794 1085 96.9 50.1 10.69 1092 97.5 50.3 10.81 5 1085 96.9 50.1 10.83 109297.5 50.2 10.75 6 1085 96.9 50.2 10.71 1092 97.5 50.4 10.69 9 1042 93.050.7 10.80 1064 95.0 50.6 10.77 12 1028 91.8 50.5 10.71 1021 91.2 50.510.53 15 1035 92.4 50.2 10.79 1014 90.5 50.3 10.62 18 1021 91.2 50.210.68 993 88.7 50.3 10.44

[0041] It is clear from the preceding data that the preparationaccording to the invention has an optimal stability in spite of thecoexistence of the chlorine-based compound with alcohol. Theconcentration of active chlorine is not remarkably reduced even afterseveral months of storage.

[0042] In order to ascertain the stability of the formulation accordingto the invention in the same experimental conditions as described in thein the patent document DD 2255, referred to in the foregoing inconnection with the prior art, the amount of active chlorine present inthe preparation according to the invention after exposure at atemperature of 50° C. for several hours was measured, obtaining theresults shown in the following table. TABLE 3 Stability vs time at 50°C. Time % cloro disponibile 16 hours 99.4 48 hours 98.7 1 week 96.3

[0043] In view of the fact that the cited prior art document reports(and considers as satisfactory) an amount of active chlorine of 95.5%after 16 hours (4.5% loss), the advantage brought by the formulationaccording to the invention is readily apparent.

[0044] Activity tests

[0045] 1. Intrinsic bactericidal activity—1^(st) series of tests

[0046] In order to ascertain the activity of the antiseptic product ofthe invention, the same preparation of the Example underwent a series ofin vitro tests at various concentrations, employing as the testmicroorganisms a strain of Staphylococcus aureus, i.e. the ATCC 6538strain, and a strain of Pseudomonas aeruginosa, i.e. the ATCC 15442strain. Both strains were supplied by the American Type CultureCollection (Maryland, USA).

[0047] Before use the bacterial strains, that had been kept frozen, weretransplanted on slants of Tryptone Soya Agar (TSA, Merck) and were keptin refrigerator at 4° C.±1° C. At the time of preparation, the bacterialstrains were transplanted three times on TSA slants and incubated at 37°C.±2° C. for 18 hours; two hours before the test the final culture wassuspended in diluent using 5 g of glass beads, and the suspension wasdiluted to obtain a count of about 1.5-5·10⁸ colony-forming units per mlof suspension (cfu/ml). The diluent employed contained 1.0 g of meatpeptone and 8.5 g of NaCl in q.s. to 1000 ml of distilled water. Inorder to count the bacterial suspensions under test, 10⁻⁶ and 10⁻⁷dilutions in the cited diluent were prepared, and from any one of saiddilutions two aliquots of 1 ml each were taken, and were transferred onPetri dishes, in 15 ml of TSA. After incubation of the dishes for 24hours at 37° C.±2° C. the colonies were counted, and the number ofcolony-forming units per ml was calculated according to the followingformula: cfu/ml=C/(n·V·d), wherein C is the sum of the colonies countedon both dishes, n=2 is the number of the dishes, V is the volume of thebacterial suspension placed on each dish and d is the dilution factorcorresponding to the dilution realized. The value thus obtained,referred to as N, represents the bacterial count of the startingsuspension.

[0048] The bactericidal activity tests, carried out in accordance withthe standard CEN/TC 216 PrEN 1040 of September 1996, consisted inplacing the bacterial cultures prepared as above into contact with theproduct under test, at 20° C.±2° C. and for a period of time of 5minutes, and in detecting the reduction of the viability of the culturesresulting from the action of the product. On the basis of preliminarytests 0.5 wt % sodium tiosulfate was chosen as the neutralizing agent(to inactivate the action of the disinfectant after the fixed contacttime). The product according to the invention has been proved at variousweight concentrations, resulting from the dilution with distilled water.The concentration of the starting product was 1.25 times the effectivetest concentration.

[0049] In each assay the sample under test (at the initialconcentration), the bacterial suspensions and the neutralizing agentwere previously stabilized at 20° C.±2° C., and 8 ml of each sampleunder test was transferred in a sterile test tube, where 1 ml ofbacterial suspension was added, prepared as above. After the fixedcontact time (5 min.) the product was inactivated by transferring 1 mlthereof in a test tube containing 8 ml of neutralizing agent and 1 ml ofdistilled water, and thoroughly stirring the mixture. After theneutralization period (5 min.±10 sec.) the number of survivingmicroorganisms in the mixture was determined in double. The count wasmade by inclusion in agar, incubating the dishes for 24 hours at 37°C.±2° C. and counting the colony-forming units in the same way asdescribed above for the count of the value of cfu/ml in the startingbacterial suspensions. The numerical value thus obtained, referred to asN_(a), represents the bacterial count in a suspension exposed for 5 min.to the product under test, the test being applied, however, on abacterial suspension 10 times more diluted than the starting solution.Therefore, the calculation of the reduction of bacterial viabilityresulting from the contact with the product under test may be made byapplying the following formula: viability reduction=N·10⁻¹/N_(a).

[0050] The following Table 4 reports the results of the tests carriedout as as above for two different effective product concentrations(corresponding to 80% and to 50% by weight, respectively) both in termsof bacterial count (values of N and N_(a)) and in terms of reduction ofthe bacterial viability. In counting the colony-forming units thevalue>3.0·10³ has been conventionally assigned when the number ofcolony-forming units was more than 300 in both dishes. On the basis ofthe above-mentioned standard, the product is considered to show abactericidal activity if, in the test conditions, the bacterialviability detected is reduced by at least a factor of 10⁵. TABLE 4 Basebactericidal activity Conc. 80% Conc. 50% N Na Viability N_(a) Viability(cfu/ml) (cfu/ml) reduct. (cfu/ml) reduct. Staphylococcus 1.7 · 10⁸ 1.4· 10² 1.2 · 10⁵ >3.0 · 10³ <5.7 · 10³ aureus ATCC 6538 Pseudomonas 1.6 ·10⁸ 1.5 · 10² 1.1 · 10⁵ >3.0 · 10³ <5.7 · 10³ aeruginosa ATCC 15442

[0051] As it can be observed from the preceding data, the productaccording to the invention appears to possess an intrinsic bactericidalactivity at a dilution of 80% by weight with respect to the formulationas produced in the Example.

[0052] 1. Intrinsic bactericidal activity—2^(nd) series of tests

[0053] A second series of in vitro tests were carded out employing thesame preparation of the Example, with a wider set of microorganisms andconditions more similar to those actually occurring in the normal use ofa disinfectant, i.e., in the presence of a high concentration of organicmatter. The organic matter employed was bovine serum albumin, in amountssuch as to obtain a final concentration of 10% by weight in each testsample. The strains employed, all supplied by the American Type CultureCollection (Maryland, USA), are listed below:

[0054]Escherichia coli ATCC 4351

[0055]Pseudomonas aeruginosa ATCC 12121

[0056]Serratia marcescens ATCC 8101

[0057]Klebsiella pneumoniae ATCC 4211

[0058]Shigella dissenteriae ATCC 9380

[0059]Proteus vulgaris ATCC 6361

[0060]Staphylococcus epidermidis ATCC 35696

[0061]Staphylococcus aureus ATCC 33501

[0062]Clostridium difficile ATCC 17858

[0063] Streptococcus pyogenes ATCC 8058

[0064] Before use the bacterial suspensions, after repeated passages,were diluted to obtain a bacterial count of about 1.0-3.9·10⁸colony-forming units per ml of suspension (cfu/ml), as shown in thefollowing table. Each bacterial strain was contacted with the productunder test and with the 10% serum albumin in the following proportions:8:1:1, respectively, for disinfectant preparation, bacterial suspensionand serum albumin. The strains were incubated at 24° C. for timeintervals of 15 sec., 30 sec. and 60 sec., and immediately after the endof the incubation period the product was inactivated by diluting it inthe ratio 1:1000 (v/v) with phosphate buffered saline (PBS), pH 7.2.After the inactivation, an inoculum from each suspension was culturedfor 18-24 hours in the following culture media: E. coli, P. aeruginosa,S. marcescens, K. pneumoniae, S. dissenteriae, P. vulgaris: Nutrientagar; S. aureus. S. epidermidis: Trypticase soy agar (Trypticase soybroth (BBL 11768) 30.0 g, agar 15.0 g, distilled water 1.0 l); S.pyogenes: Rabbit blood agar; C. difficile: Beef liver medium foranaerobes (ATCC culture medium 38). The temperature conditions were asfollows: E. coli, K. pneumoniae, S. dissenteriae, P. vulgaris, S.aureus, S. epidermidis, C. difficile (+anaerobiosis), S. pyogenes. 37°C.; P. aeruginosa, S. marcescens: 26° C.

[0065] At the end of the culture period the colonies formed werecounted, and the number of colony-forming units per ml (cfu/ml) wascalculated according to the same formula given in the previous section.The controls employed in the test were the same bacterial suspensions,treated with PBS (pH 7.2) instead of the disinfectant under test, in thesame conditions and for the same times. The following Table 5 reportsthe results of the tests carried out as described above. TABLE 5 Basebactericidal activity inoculum 15 sec. 30 sec. 60 sec. (cfu/ml) (cfu/ml)(cfu/ml). (cfu/ml) C. difficile 1.0 · 10⁸ <100 <100 <100 E. coli 1.6 ·10⁸ <100 <100 <100 S. pyogenes 2.2 · 10⁸ <100 <100 <100 P. aeruginosa3.9 · 10⁸ 0.32 · 10³ <100 <100 S. marcescens 2.4 · 10⁸ 0.68 · 10³ <100<100 K. pneumoniae 1.1 · 10⁸ 0.23 · 10³ <100 <100 S. dissenteriae 1.7 ·10⁸ <100 <100 <100 P. vulgaris 1.2 · 10⁸ <100 <100 <100 S. epidermidis2.0 · 10⁸  1.4 · 10² <100 <100 S. aureus 1.0 · 10⁸ 0.22 · 10³ <100 <100

[0066] The results reported in the above table show a virtually completeinhibition (values below 100 cfu/ml) after 30 seconds of incubation forall of the bacterial strains tested. Even after 15 seconds of contactbetween the microorganisms and the product of the invention thebacterial counts obtained are, for the totality of the strains tested,equivalent to a reduction by a factor of at least 10⁵, this being thelimit, as pointed out in the previous section, for establishing anantimicrobial activity.

[0067] 2. Bactericidal activity in the conditions of use for the hands

[0068] Tests similar to those reported as the 1^(st) series of tests forintrinsic bactericidal activity were carried out according to thestandard CEN prEN 12054 of July 1995, relevant to the hands treatmentbefore surgery (i.e. surgical handrub). The strains Pseudomonasaeruginosa ATCC 15442, Staphylococcus aureus ATCC 6538, Enterococcusfaecium ATCC 10541 and Escherichia coli K12 NCTC 10538 were used, all ofthem supplied by the American Type Culture Collection (Maryland, USA).

[0069] The preparation of the starting bacterial suspensions and thecalculation of the number of colony-forming units (N) of said bacterialsuspensions were the same as in the previous test, with the onlydifference that the bacterial count in the initial suspensions was about1- 3·10⁸ cfu/ml. Also the choice of the neutralizing agent and thetemperature of performance of the test (20° C.±2° C.) were the same asin the previous test, but the effective concentration of product usedwas of 90% by weight, and the measures were taken for two differentcontact times, i.e. 2 minutes and 5 minutes.

[0070] For each bacterial strain a test tube was prepared containing 9ml of product under test and 1 ml of bacterial suspension, and at theend of each contact time 1 ml of the mixture was transferred in a testtube containing 8 ml of neutralizing agent and 1 ml of distilled water.After 5 minutes of neutralization the mixture was vortexed and the countwas carried out, in double, with the same procedure as the previoustest, obtaining the values of cfu/ml after exposure to the product(N_(a)) for the fixed periods of time. In the cfu count, thevalue<3,0·10² has been conventionally assigned when the number ofcolony-forming units was lower than 300 in both dishes. In this case thematerial under test is considered to be bactericidal when it causes, foreach strain, a reduction of the bacterial count from 1-3·10⁸ cfu/ml tono more than 3·10² cfu/ml after 5 minutes of contact. TABLE 6Bactericidal activity in the conditions of use on the hands 90%Concentration after 3 min. after 5 min. N colony N_(a) colony N_(a)(cfu/ml) count (cfu/ml) count (cfu/ml) Staphylococcus 1.49 · 10⁸ 29-272.8 · 10² 18-13 1.55 · 10² aureus ATCC 6538 Pseudomonas 1.65 · 10⁸ 30-362.8 · 10² 14-19 1.6 · 10² aeruginosa ATCC 15442 Escherichia 1.80 · 10⁸0-0 <3.0 · 10² 0-0 <3.0 · 10² coli K12 NCTC 10538 Enterococcus 1.99 ·10⁸ 14-21 1.71 · 10² 0-0 <3.0 · 10² faecium ATCC 10541

[0071] As it is noted from the data in the above table, the productaccording to the invention appears to possess the desired bactericidalactivity also after a time of exposure of 3 minutes only.

[0072] 3. Practical test for bactericidal activity on thehands—comparison with 60% isopropanol

[0073] By using a strain of Escherichia coli K12 NCTC 0538 obtained fromthe Sierotherapic Center of Milan, Italy, some tests were carried out toascertain the antiseptic and hygienizing activity of the product of theExample in the normal use conditions for hands washing. For the test 12voluntaries of both sexes were employed, selected on the basis of theperfectly intact status of the skin, the good general health conditionsand the absence of ongoing pathologies and pharmacological treatments.

[0074]Escherichia coli was cultured in two test tubes containing 5 ml ofTSB (Tryptone Soya Broth, Merck) for 24 hours at 37° C.±2° C. and thecultures were inoculated in two bottles containing 1 l of TSB each, andincubated for 24 hours at 37° C.±2° C. This contaminating liquidcontains about 2·10⁸-2·10⁹ cfu/ml, the number of colonies having beendetermined by carrying out the count in double by inclusion in agar,incubating the Petri dishes at 37° C.±1° C. for 24 hours.

[0075] At the beginning of the test, all of the voluntaries washed theirhands with a linseed oil soap to remove the dirt naturally present, andthen dried their hands with disposable paper towels. Each voluntarydipped then his fingertips in the bacterial suspension for 5 seconds,and dried them in air for 3 minutes. Immediately after, each voluntarydipped for 1 minute his contaminated fingertips of both handsrespectively in two Petri dishes containing 10 ml of TSB, so as to allowthe evaluation of the number of microorganisms released before thetreatment. From the fluid thus obtained decimal dilutions 10⁻³-10⁻⁴ wereprepared, and for each dilution 0.1 ml were spread on the surface ofPetri dishes containing TSA, by using glass beads. The dishes were thenincubated at 37° C.±2° C. for 24 hours, and the number of colonies foreach dilution was counted, calculating the arithmetic mean thereof. Thenumber of colonies counted was then transformed in the decimallogarithmic value, and is referred to as Log x (basal contaminationvalues).

[0076] Immediately after the determination of the initial values, andwithout recontamination of the hands, 6 of the 12 vuntaries were treatedwith a comparative solution of isopropyl alcohol 60% v/v and the other 6were treated with the solution according to the invention. The first 6voluntaries washed both hands with 3 ml of isopropyl alcohol for 30seconds and then repeated the operation with other 3 ml of alcohol for60 seconds. They finally rinsed their hands for 5 seconds with runningwater. The other 6 voluntaries used a surgical gauze soaked with 4 ml ofthe product under test, cleaning their hands for 1 minute, and left thendry their hands in the air for 2 minutes.

[0077] The voluntaries of both groups dipped then for 1 minute theirfingertips of both hands in two Petri dishes respectively, and the sameprocedure reported above was repeated for the determination of thenumber of bacterial colonies originated. The values of the decimallogarithms thus obtained are indicated as Log y (post-treatment values).The logarithmic values of the reduction of the bacterial count from thebasal value to the post-treatment value are referred to as Log z.

[0078] In a second stage of the test the whole procedure was repeatedwith the same voluntaries, after having exchanged the two products undertest. Therefore, for each subject the following Table 7 shows either thevalues relevant to the treatment with isopropanol and the valuesrelevant to the treatment with the preparation of the invention. TABLE 7Practical test of bactericidal activity on the hands Voluntary 60%isopropanol Product of the invention No. Log x Log y Log z Log x Log yLog z 1 6.49 4.11 2.37 6.37 1.75 4.62 2 6.41 4.20 2.21 6.32 1.92 4.40 36.33 4.35 1.98 6.30 1.83 4.47 4 6.38 3.78 2.60 6.33 1.45 4.88 5 6.363.87 2.49 6.48 1.91 4.57 6 6.42 4.07 2.35 6.43 1.82 4.61 7 6.44 4.272.17 6.36 1.80 4.56 8 6.38 4.38 2.00 6.41 1.88 4.53 9 6.26 4.28 1.986.32 1.86 4.46 10 6.24 4.00 2.64 6.32 1.67 4.65 11 6.22 4.49 2.73 6.201.52 4.68 12 6.37 3.46 2.91 6.35 1.91 4.44

[0079] It is evident from the above results that the formulation ofchloramine-T in 50% isopropanol solution according to the invention hasan antiseptic activity remarkably higher than that of the comparisondisinfectant product, i.e. 60% isopropanol.

[0080] Cutaneous irritation tests

[0081] In order to ascertain the cutaneous tolerability of the productof the Example, a test on 3 male New Zealand albino rabbits was carriedout. The rabbits, weighing 2.5-3.5 kg, had been randomly selected amonga certain number of animals that had been previously kept in quarantinefor a week and then underwent, before the test, a thorough examinationto ascertain their suitability for the test. The back and the sides ofthe animals were shaved 24 hours before starting the test, on an area ofabout 240 cm², and an area of about 20 cm² on the right side has beenused for applying the sample under test, while the left side, untreated,served as a control. 0.5 ml of the product were applied by means of asquare piece of surgical gauze directly on the skin, and the gauze wasfixed with a plaster. The square gauze was further fixed with anocclusive hypoallergenic adhesive tape, and the whole trunk of theanimal was protected by an elastic medication.

[0082] About 4 hours after the application, the medications and theadhesives were removed, and the skin was cleaned from the excess sample.The cutaneous reactions of the animals were evaluated one hour after theremoval of the gauze. For the evaluation of the cutaneous reactions thefollowing score scales were used:

[0083] Erythema and eschar formation: 0=absence of erythema; 1=lighterythema (hardly visible); 2=well-visible erythema; 3=erythema frommoderate to severe; 4=severe erythema (beet red) or eschar formation(deep lesions). Oedema formation: 0=absence of oedema; 1=very lightoedema (hardly visible); 2=light oedema (margins of the inflated zonewell defined); 3=moderate oedema (margins projecting by about 1 mm);4=strong oedema (margins projecting by more than 1 mm, inflationextended beyond the area of application).

[0084] The values of the cutaneous reactions found in each of the threerabbits after 60 minutes, 24 hours, 48 hours and 72 hours from theremoval of the gauze with the product under test, evaluated by means ofthe above score scales, have always been equal to 0, indicating thetotal absence of any erythematous and/or oedematous reaction.

[0085] The present invention has been disclosed with particularreference to some specific embodiments thereof, but it should beunderstood that modifications and changes may be made by the personsskilled in the art without departing from the scope of the invention asdefined in the appended claims.

1. A disinfecting preparation containing chlorine in alcohol solutioncomprising, in water, chloramine-T or chloramine-B and one or morealiphatic alcohols having up to 12 carbon atoms, said solution having apH not lower than 8.5.
 2. A disinfecting preparation according to claim1 comprising, in addition, a buffer suitable to keep the pH at a valuenot below 8.5.
 3. The disinfecting preparation according to claims 1 or2, wherein said chloramine-T or said chloramine-B is present at aconcentration corresponding to 1000-2500 ppm of available chlorine. 4.The disinfecting preparation according to any one of claims 1-3, whereinsaid one or more aliphatic alcohols are chosen from ethanol, n-propanoland isopropanol.
 5. The disinfecting preparation according to claim 4,wherein said ethanol and/or said n-propanol and/or said isopropanol arepresent in an overall concentration comprised between 40 and 70% byweight.
 6. A disinfecting preparation according to any one of claims2-5, wherein said buffer is borate buffer.
 7. The disinfectingpreparation according to claim 6, having the following parameters:chloramine-T or chloramine-B 1000-2500 ppm of available chlorine ethanolor isopropanol 50-60% by weight pH >8.5. boric acid 0.153-0.155% byweight sodium hydroxide 0.0396-0.064% by weight water q.s. to 100%.


8. The disinfecting preparation according to claim 7, wherein saidchloramine is chloramine-T.
 9. The disinfecting preparation according toclaim 8, wherein the concentration of said chloramine-T corresponds to1000-2500 ppm of available chlorine.
 10. The disinfecting preparationaccording to any one of claims 7-9, wherein said alcohol is isopropanol.11. The disinfecting preparation according to claim 10, wherein theconcentration of said isopropanol is 50% by weight.
 12. The disinfectingpreparation according to any one of claims 7-11, wherein said pH iscomprised between 10.4 and 10.9.